Preliminary Study Of Self-assembled Nanocarrier Systems Based On Amphiphilic Chitosan Derivative For Oral Vaccine Delivery

In China, intensive carpfarming has become a key industry in recent decades. Massmortality will occur if Aeromonas hydrophila are introducedinto the farm, causing great financial losses. It isnecessary to induce sufficient protective immune responsesin carps. In the past decade, many strategies have been developed toenhance oral vaccinedelivery. Among theseapproaches, nanoparticulate systems have attracted specialinterest because nanoparticles are able to protect activeagents from degradation, improve macromolecules transmucosal transport and provide controlled-releaseproperties for loaded drugs. Moreover, mucoadhesiveproperties also play an important role in oral drugdelivery systems by prolonging the residence time of drugcarriers and also increasing the intimacy of contactbetween drug and mucous membrane at the absorptionsites; therefore, enhancing the permeability and reducingthe degradation of drugs.Oleoyl-carboxymethyl chitosan (OCMCS) based on chitosan with different molecular weights (50kDa,170kDa and820kDa) was synthesized and could be soluble at neutral pH.OCMCSnanoparticleswere prepared using coacervation process,which demonstrated particle size of150-350nm, zeta potential of10-20mV and an almost spherical shape.The OCMCS nanoparticles showed low erythrocyte membrane-damaging effect. The MTT survival assay indicated no significant cytotoxicity.In vivo toxicity assays were performed via histopathological evaluation, and no specific anatomical pathological changes or tissue damage was observed in the tissues of carps(Cyprinus carpio). These results demonstrated that OCMCS nanoparticles could be applied as safe carriers for the oral administration of drugs.FD4,a model macromolecule, was encapsulated in OCMCS nanoparticles.FD4-OCMCS nanoparticles were found to be adsorbedonto the excised carp intestinal mucosa, the extent of adsorption increased with increasing chitosan molecular weight.In comparison to FD4solution, OCMCS nanoparticles promoted FD4transport through excised carp intestinal mucosa by3.26-6.52folds, which were observed via fluorescence microscope.The OCMCS nanoparticulate systems that interacted with the Caco-2cells decreased the transepithelial electric resistance (TEER) and induced increasing the apparent permeability coefficient Papp)of FD4by3.61-6.32folds. The improvement of mucoadhesive ability and permeability enable the OCMCS nanoparticles suitable carriers for the intestinal absorption ofmacromolecular drugs.The extent of tissue distribution and retention following oral administration of FITC-OCMCS nanoparticles to carps was analyzed for3days.After3days, the nanoparticles remained detectable in the muscle, heart, kidney, liver, intestine, and spleen. The results showed that34.32%of the particles were localized in the liver,18.79%in the kidney, and17.36%in the heart. The lowest percentage was observed in the muscle. The results indicated that OCMCS nanoparticles were internalizedvia the intestinal mucosa performed good tissue distribution after orally fed in carps.Extracellular products (ECPs) of Aeromonas hydrophila as microbial antigen was efficiently loadedto form OCMCS-ECPs nanoparticles and shown to be sustained release in PBS.Significantly higher (P<0.05) antigen-specific antibodies were detected in serum after orally immunized with OCMCS-ECPs nanoparticles than that immunized with ECPs alone and non-immunized in control group in carps. Thepermeation of ECPs via the mucouslayerwas enhanced, which might be attributed to the mucoadhesion and permeation enhancement effect of OCMCS nanoparticles.Nanoparticles made of OCMCS and hyaluronic acid (HA) were obtained as novel potential carriers for gene delivery.An N/P ratio of5and OCMCS/HA weight ratio of4were the optimal conditions leading to the smallest, positive charged and monodispersed nanoparticles.OCMCS-HA/DNA nanoparticles showed higher in vitro DNA release rates and increased cellular uptake by Caco-2cells due to the HA involved in nanoparticles.The transfection efficiency of OCMCS-HA/DNA NPs was5-fold higher than OCMCS/DNA NPs; however, it decreased significantly in the presence of excess free HA.The results indicated that OCMCS-HA NPs were internalized byfluid endocytosis and that this endocytic process was mediated by the hyaluronan receptor CD44. The data obtained in the present research gave evidence of the potential of OCMCS-HA NPs for the targeting and further transfer of genes to the epithelial cells.These results obtained in the present work implied that OCMCS nanoparticles had great potential to be applied as safe and effectiveoral vaccine carriers at neutral pH.